A. Diseases in Shellfish (oysters, clams, and other bivalves)
Shellfish, particularly oysters, are universally recognized as important sources of commercially valuable food and as organisms that play important roles in the aquatic ecosystem as part of the food chain and in reducing the turbidity of water through filtration. Unfortunately, protozoan, bacterial, fungal and viral epizootic diseases are destroying massive numbers of natural and cultivated stocks of oysters and other shellfish in coastal areas of the United States. A clear example of the serious impact of shellfish diseases is the enormous decline in oyster production from the Chesapeake Bay. Oyster production has plummeted from a high of 2.5 million bushels harvested annually in the early 1980's to less than 1% of this level in the past few years.
Protozoan infections is a primary cause of mass mortality of the eastern oyster Crassostrea virginica along the Gulf of Mexico and Atlantic coasts. The major disease is "Dermo," caused by the endoparasitic protozoan Perkinsus marinus. This disease, for which there is no known remedy, has resulted in a critical reduction of existing populations and is a major cause of the collapse of the oyster industry in the Chesapeake Bay. The range of this parasite has now extended into low salinity areas of Chesapeake Bay tributaries that are sources of oyster seed stock. Additionally, the parasite has been detected in North Atlantic waters from Delaware Bay to Maine that were previously disease free and thought to be uninfected due to cold water conditions.
Other Perkinsus species have been detected in mollusks around the world and cause mass mortalities in commercially important shellfish from Australia and Europe. In addition to P. marinus, other pathogenic species include P. olseni in the abalone in Australia and P. atlanticus in the European clam.
The transplantation of brood and seed stocks between countries has become a frequently used alternative to raising native shellfish. However, this practice can also lead to the spread of disease and the destruction of native stocks because of the lack of appropriate diagnostic tests. Frequently, natural resource managers seek to introduce non-indigenous oysters having desirable characteristics to their aquatic jurisdiction. However, if the introduced species carries Dermo or other infectious diseases the consequences can be devastating.
B. Currently Available Shellfish Dermo Disease Detection Methods
The continuing decline of oyster stocks as a result of Dermo and other diseases has created a demand for new technologies to efficiently detect and monitor these diseases in indigenous and transplanted oysters. The most significant obstacle to developing effective treatment and management strategies for controlling P. marinus infections is the lack of a sensitive assay that would allow for both the detection of P. marinus at low infection levels and discrimination between putative geographic subpopulations of P. marinus as well as other Perkinsus species. There is a need for sensitive and specific diagnostic assays for P. marinus to detect, for example, cryptic infections in oyster seed-stocks, latent infections in overwintering oyster populations, the onset of infection in oyster larvae and spat, the presence of P. marinus in other marine organisms that may serve as secondary vectors or reservoirs, and the genetic structure of parasite field populations.
The life cycle of P. marinus within the host consists of an intracellular vegetative state (trophozoite) which proliferates by multiple fusion and/or budding. Mature trophozoites enlarge to become prezoosporangia, which upon entering the water column sporulate to release large numbers of biflagellated zoospores. These motile zoospores presumably give rise to trophozoites once they infect oyster tissue, but the mechanism of infection is unknown. With most prior art detection methods only trophozoites can be detected in most host tissues but not the other stages. It would be desirable to have an assay that is sensitive enough to detect any P. marinus life stage present in a sample.
Histology was the first technique used for diagnosis of Perkinsus marinus. The fluid thioglycollate media (FTM) assay (Ray,1952,1966) which has been the routine method for Perkinsus species diagnosis was adopted because it was inexpensive and simple to perform. In the FTM assay oyster tissue is incubated with antibiotic-fortified medium under conditions in which parasites at the trophozoite stage enlarge into hypnospores. These stain with Lugol's iodine solution for visualization of the parasite as a blue-dark sphere (Ray, 1966). The FTM assay relies on the enlargement of the trophozoites into hypnospores in fluid thioglycollate medium, a feature shared by all Perkinsus species and so does not distinguish between them. Hence this assay is not species specific. Consequently, most studies on Perkinsus from bivalves refer to them as Perkinsus species because no specific identification is possible. In addition, this assay is only able to detect one stage in the lifecycle of these parasites and takes between 4 and 7 days to complete. Hence, effective diagnosis in terms of sensitivity, species-specificity, and rapidity are needed for appropriate management of bivalve resources.
Antibody-based assays for the detection of P. marinus proteins in oyster tissues have recently been used with mixed success due to lack of sensitivity (Choi et al., 1991; Dungan and Roberson, 1993). These antibodies were raised against only one life stage of this parasite. Consequently the lack of sensitivity may be due to changes in epitope expression by the parasite at different life cycle stages. Also, a general feature of parasites is their ability to modify their epitope expression over time making an antibody-based assay unreliable. Because of these disadvantages, this technique never became established as a routine diagnostic assay for Perkinsus.
The sensitivity of PCR for detection of trace quantities of foreign DNAs in heterogenous samples has made this technology an ideal choice for identifying infectious agents and has been used with great success to screen protozoan pathogens in aquaculture (Cai et al., 1992; Stokes and Burreson, 1995). Different gene regions have been used as PCR targets. The ability of a PCR assay targeting DNA to distinguish between genetically related species and subspecies depends on the correct choice of a gene target. Fong et al. (1993) suggested the use of the small subunit of the rRNA gene of Perkinsus to design probes for this parasite, however this region cannot be used as a PCR target because the high degree of sequence identity that exists in homologous genes among between this parasite and its host.
The introduction and transplantation of shellfish has contributed to the spread of disease. The Working Group on Diseases of the International Council for the Exploration of the Seas (ICES) has established criteria for the introduction of exotic species as well as for transferred species. These criteria require periodic inspection and testing of the material using state of the art techniques before the mass transplantation and during quarantine. In addition, a significant obstacle to developing effective treatment and management strategies for controlling P. marinus infections in oysters is identifying when exactly an infection begins and the source of the pathogen. The only diagnostic technique routinely used up to this point has been the FTM assay which, as described, lacks the necessary requirements of sensitivity and specificity in detection of the parasite in order to help guarantee disease-free oysters.
There is a strong need, therefore, for a diagnostic assay that is (1) sensitive enough to detect the presence of the various species of Perkinsus at low levels, and (2) specific enough to discriminate between putative geographic races or strains of P. marinus and between the various species of Perkinsus, and (3) that can be completed rapidly enough to provide resource managers with timely information about the disease status of oyster populations, especially of oysters proposed for introduction from distant sources.